Structure and Function of the Trypanosome Flagellar Membrane A number of closely related calcium-binding proteins are found in the flagella of the protozoan parasites Trypanosoma cruzi and Trypanosoma brucei: a 24 kDa flagellar calcium binding protein (FCaBP) of T. cruzi and a family of 24 kDa (Tb-24), 25 kDa (Tb-17), and 44 kDa (Tb-44) calfagin proteins of Trypanosoma brucei. These proteins are modified by the addition of myristate and palmitate at their amino termini, which mediate localization to the flagellar membrane. Various properties of the calflagins have been determined during the previous funding period: (i) they bind calcium and undergo calcium-induced changes in conformation, (ii) their localization to the flagellar membrane is dependent on the concentration of membrane sterols, (iii) they appear to function through the calcium-dependent binding and presumed regulation of partner proteins, (iv) they have unique three-dimensional structures that are similar to but distinct from calmodulin and (v) they are components of and/or substrates for the intraflagellar transport (IFT) apparatus. Additional, related discoveries arising from from this work include the following: (i) the flagellar membrane is chemically distinct from the pellicular membrane and shows a high degree of liquid order and enrichment in lipid rafts, (ii) a flagellar DnaJ (hsp40) protein is involved in the biogenesis of the cytostome and (iii) sphingolipid synthesis is essential for normal cytokinesis and organelle segregation in T. brucei. Our work on FCaBP and the calflagins has begun to illuminate the functions of these proteins in flagellar function and biogenesis and has revealed the flagellar membrane as a novel platform for the recruitment of dually acylated proteins and participation in IFT. Information from colleagues indicate that these basic properties may be common to all ciliary structures (flagella and sensory cilia) and thus the trypanosome may be a powerful model system for the determination of ciliary function generally. We propose to extend our studies of these proteins specifically and the flagellar membrane generally in a five-year research effort having the following Specific Aims: (1) To define the molecular determinants of flagellar protein targeting. (2) To examine the interaction between intraflagellar transport and flagellar membrane trafficking. (3) To determine the lipid composition of the flagellar membrane and its contribution to protein targeting. PUBLIC HEALTH RELEVANCE: Over 200 million people are at risk of infection Trypanosoma cruzi, the parasitic agent of Chagas disease and Trypanosoma brucei, the agent of African sleeping sickness. The drugs available for treating trypanosome infections are not very effective and suffer from poor efficacy and high toxicity. Our studies are aimed at identifying novel process of trypanosome cell biology that can be exploited for the development of new drugs for Chagas disease and African sleeping sickness. Further, our proposed studies of flagellar- ciliary membrane targeting are relevant to many human diseases, collectively known as the "ciliopathies." The ciliopathies cause kidney disease, diabetes, cancer, developmental disorders, and blindness, and some of these human diseases are likely to be caused by defects in the processes we are studying in this research project.